Riboswitch RNAs represent an important regulatory mechanism in bacteria. RNAs of this type consist of complex elements positioned in the leader region of a transcript, upstream of the regulated coding sequence(s). These RNA elements directly sense a physiological signal that induces a structural change in the RNA, resulting in an effect on downstream gene expression. This project will focus primarily on two classes of S-adenosylmethionine (SAM)-binding riboswitch RNAs, the S box and the SMK box, with additional efforts on the lysine-binding L box, the thiamine pyrophosphate-binding Thi box, and RNA thermosensors that respond to changes in temperature. The first major goal of the project is to investigate the molecular basis for specific ligand recognition by multiple riboswitch RNAs, and the features responsible for differential SAM sensitivity in natural variants of the S box riboswitch. These efforts will also include using the information obtained to engineer novel classes of riboswitches with the goal of testing the predictive power of these analyses. The second major goal is to investigate the structural and functional differences between riboswitches that operate at the transcriptional and translational levels, to test the hypothesis that translational riboswitches (like the SMK box) have the potential to operate reversibly in vivo, allowing multiple regulatory decisions within the lifetime of a single RNA transcript. These studies will include detailed analysis of the ligand-free form of the SMK box RNA and the transition between the ligand-free and ligand-bound forms. The third major goal involves analysis of the interplay between riboswitch elements and other regulatory mechanisms, using the Bacillus subtilis metK gene, encoding SAM synthetase, as an example. Overall, this project will provide basic information about novel RNA-based mechanisms of gene regulation, and will also provide insight into metabolic regulation in pathogenic organisms that use these mechanisms. Gram- positive pathogens generally use regulatory mechanisms closely related to those found in Bacillus subtilis, the model organism for this work. Expression of determinants for pathogenicity are often regulated in response to physiological signals, and understanding how the cell monitors these signals is important for understanding bacterial virulence. It is also likely that many new riboswitch-like mechanisms remain to be uncovered, and the proposed work will provide important tools for investigation of these mechanisms, and predicting how they function within the cell. PUBLIC HEALTH RELEVANCE: RNA-mediated regulation has recently emerged as a central player in all organisms. This study is directed toward the analysis of metabolite-binding riboswitches, a class of regulatory RNAs that directly sense a physiological signal and transmit that information to the gene expression machinery via an RNA structural rearrangement. This mechanism is widely used in bacteria, including in a number of important pathogens, and has also been identified in archaea and eukaryotes. The goal of this project is to investigate the molecular mechanisms underlying RNA-mediated ligand recognition and gene regulation in response to modulation of RNA structure.